The present invention relates generally to biologically active compounds and more specifically to compounds and peptides which are amphipathic, i.e., have both hydrophilic and hydrophobic portions. Specifically, the invention relates to improved methods for the delivery and presentation of amphipathic peptides in association with liposomes for both diagnostic and therapeutic uses.
Of particular interest to the present invention are the biologically active amphipathic peptides which are members of the family of peptide compounds including vasoactive intestinal peptide (VIP) and growth hormone releasing factor (GRF). More specifically, the invention relates to improved therapeutic methods for delivering peptides in the VIP/GRF family of peptides to targeted tissues through use of improved liposome compositions comprising a member of the VIP/GRF family of peptides and biologically active analogues thereof.
VIP is a 28-amino acid neuropeptide which is known to display a broad profile of biological actions and to activate multiple signal transducing pathways. See, Said. Peptides 5 (Suppl. 1):149-150 (1984) and Paul and Ebadi, Neurochem. Int. 23:197-214 (1993). A Schiff-Edmundson projection of VIP as a xcfx80-helix reveals segregation of apolar and polar residues onto the opposite faces of the helix and that this amphipathic character is also evident when VIP is modeled as a distorted xcex1-helix, which is reported in Musso, et al., Biochemistry 27:8147-8181 (1988). A correlation between the helixforming tendency of VIP analogues and their biological activity is described in Bodan et al., Bioorgan. Chem. 3:133-140 (1974). In pure water, the spectral characteristics of VIP are consistent with those of a random coil. However, organic solvents and anionic lipids induce helical-information in the molecule. See, Robinson et al., Biopolymers 21:1217-1228 (1983); Hamed, et al., Biopolymers 22 :1003-1021 (1983); and Bodanszky, et al,Bioorganic Chem. 3:133-140 (1974).
Short peptides capable of forming amphipathic helices are known to bind and penetrate lipid bilayers. See, Kaiser and Kezdy, Ann. Rev. Biophys. Biophysical Chem. 15:561-581 (1987) and Sansom, Prog. Biophys. Molec. Biol. 55:139-235 (1991). Examples include model peptides like (LKKLLKL-), which are disclosed in DeGrado and Lear, J. Am. Chem. Soc. 107:7684-7689 (1985), and the 26-residue bee venom peptide, melittin, disclosed in Watata and Gwozdzinski, Chem-Biol. Interactions 82:135-149 (1992). Possible mechanisms for the binding include alignment of peptide monomers parallel to the surface of the bilayer mediated by electrostatic interactions between polar amino acids and phospholipid head groups, and insertion of peptide aggregates into the apolar bilayer core, stabilized in part, by the hydrophobic effect. See. Sansom, Prog. Biophys. Molec. Biol. 55:139-235 (1991).
VIP belongs to a family of homologous peptides, other members of which include peptide histidine isoleucine (PHI), peptide histidine methionine (PHM), growth hormone releasing factor (GRF), pituitary adenylate cyclase activating peptide (PACAP), secretin and glucagon. Like VIP, the other members of the VIP/GRF family of peptides, and biologically active analogues thereof, can form amphipathic helices capable of binding lipid bilayers. The biological action of members of the VIP/GRF family of peptides are believed to be mediated by protein receptors expressed on the cell surface and intracellular receptors and it has recently been demonstrated that calmodulin is likely to be the intracellular receptor for VIP [Stallwood, et al., J. Bio. Chem. 267:19617-19621 (1992); and Stallwood, et al., FASEB J. 7:1054 (1993)].
A major factor limiting in vivo administration of VIP has been its reduced bioavailability at target tissues mostly because of proteolytic degradation, hydrolysis, and/or a multiplicity of conformations adopted by the peptide. It has been speculated that intracellular delivery of VIP alone and/or VIP-calmodulin mixtures could bypass the requirement for cell-surface binding of the peptide and thus enhance the biological actions of the peptide. Provision of the peptides expressed in and on liposomes would possibly permit intracellular delivery, since lipid bilayers of liposomes are known to fuse with the plasma membrane of cells and deliver entrapped contents into the intracellular compartment.
Characterization of the structure and properties of liposomes led to many proposed uses for the vesicle as vehicles to effect targeted drug delivery, most of which failed to materialize for any of a number of various reasons. Most prominently, the therapeutic parenteral use of conventional liposomes was found to be limited because of rapid uptake into the reticuloendothelial system by mononuclear phagocytic cells [Gregoriadias and Ryman, Eur. J. Biochem. 27:485-491 (1972); Beaumier, and Hwang, Biochem. Biophys. Acta 731:23-30 (1983)]. Uptake by this particular cell type is advantageous under the limited conditions wherein the targeted cell or tissue itself is part of the reticuloendothelial system, but uptake by phagocytic cells generally leads to degradation of compounds to be delivered, thereby posing a serious drawback to delivering a compound to other cell or tissue types.
In attempts to overcome problems inherent to liposome drug delivery, research turned to several approaches including identification of compounds which would be released back into the blood following liposome uptake by the-reticuloendothelial system, alternatives to intravenous liposome administration, and use of various compounds, for example, cholesterol, to increase liposome stability in the bloodstream [Kirby, et al., Biochem. J. 186:591-598 (1980); Hwang, in Liposomes from biophysics to therapeutics, Ostro (ed.) Marcel Decker: New York (1987) pp. 109-156; Beaumier, et al., Res. Comm. Chem. Pathol. Pharmacol. 39:227-232 (1983)]. Still other investigations examined various lipid compositions to form the liposome bilayer which more closely mimic the naturally occurring bilayer of red blood cell. Such efforts led to increased liposome half-life in circulation [Allen and Chonn, FEBS Lett. 223:42-46 (1987); Gabizon and Papahadjopoulos, Proc. Natl. Acad. Sci. (USA) 85:6949-6953 (1988)].
PCT Publication WO 95/27496 and Gao, et al., Life Science 54:247-252 (1994) describe the use of liposomes for delivery of VIP in comparison to its delivery in aqueous solution. Encapsulation of VIP in liposomes was found to protect the peptide from proteolytic degradation and to significantly enhance the ability of VIP and to effect a decrease in mean arterial pressure in comparison to VIP in aqueous solution in hypertensive hamsters. Liposome-associated VIP was found to significantly decrease mean arterial blood pressure for a period of approximately 12 minutes, with lowest blood pressure observed almost 5 minutes after initial administration. The publication also demonstrated binding of VIP in aqueous solution to liposomes and penetration of the peptide into the liposome bilayer. It was speculated that binding of VIP to liposomes might prevent loss of peptide activity either by partitioning of the peptide into the liposome membrane, stabilizing the peptide against proteolysis, or restricting the peptide in a biologically active conformation. Whatever the reason, encapsulation of VIP in liposomes enhanced in vivo biological activity of the peptide by both prolonging the effect and increasing the magnitude of the effect in lowering blood pressure of hypertensive hamsters. Nevertheless, there remains a desire in the art to provide further improvements in the therapeutic and diagnostic delivery of biologically active peptides such as VIP.
Of interest to the present invention is the observation of increased half-life of circulating protein through conjugation of the protein to a water soluble polymer [Nucci, et al., Adv. Drug Del. Rev. 6:133-151 (1991); Woodle, et al., Proc. Inten. Symp. Control. Rel. Bioacr. Mater. 17:77-78 (1990)]. This observation led to the development of sterically stabilized liposomes (SSL) (also known as xe2x80x9cPEG-liposomesxe2x80x9d) as an improved drug delivery system which has significantly minimized the occurrence of rapid clearance of liposomes from circulation. [lasic and Martin, Stealth Liposomes, CRC Press, Inc., Boca Raton, Fla. (1995)]. SSL are polymer-coated liposomes, wherein the polymer, preferably polyethylene glycol (PEG), is covalently conjugated to one of the phospholipids and provides a hydrophilic cloud outside the vesicle bilayer. This steric barrier delays the recognition by opsonins, allowing SSL to remain in circulation much longer than conventional liposomes [Lasic and Martin, Stealth Liposomes, CRC Press, Inc., Boca Raton, Fla. (1995); Woodle, et al, Biochem. Biophys. Acta 1105:193-200 (1992); Litzinger, et al., Biochem. Biophys. Acta 1190:99-107 (1994); Bedu Addo, et al., Pharm. Res. 13:718-724 (1996)]and increases the pharmacological efficacy of encapsulated agents, as demonstrated for some chemotherapeutic and anti-infectious drugs [Lasic and Martin, Stealth Liposomes, CRC Press, Inc. Boca Raton, Fla. (1995)]. Studies in this area have demonstrated that different factors affect circulation half-life of SSL, and ideally, the mean vesicle diameter should be under 200 nm, with PEG at a molecular weight of approximately 2,000 Da at a concentration of 5% (9-12) [Lasic and Martin, Stealth Liposomes, CRC Press, Inc., Boca Raton, Fla. (1995); Woodle, et al., Biochem. Biophys. Acta 1105:193-200 (1992); Litzinger, et al, Biochem. Biophys. Acta 1190:99-107 (1994); Bedu Addo, et al., Pharm. Res. 13:718-724 (1996)]. Preparation of SSL having these physical properties and including a bioactive compound, however, is not without complications as activity of the associated compound can be lost in preparation of SSL having desirably characteristics. This is particularly the case where an extrusion process is used to obtain small size liposomes with a narrow particle size distribution. For reasons which are not completely understood, such extrusion methods substantially reduce the biological activity peptide components associated with the liposomes. Accordingly, there remains a desire for improved liposome compositions which are sterically stable but which maintain the biological activity of associated peptide agents.
Also of interest to the present invention is the disclosure of PCT Publication WO 93/20802 which relates to multilamellar liposomes useful for enhancement of organ imaging with acoustics (ultrasound). The publication describes various liposome compositions ranging in size from 0.8 to 10 microns including a tissue specific ligand, such as an antibody, antibody fragment or a drug incorporated into the lipid bilayer, in order to facilitate tissue specific targeting. The oligolamellar liposomes are prepared by processes such as lyophilization, repeated freeze-thaw, or modified double emulsion techniques to produce internally separated bilayers. Preferred liposomes are said to range from 1.0 to 3.0 microns in diameter. It has thus far been more difficult to produce liposomes which are readily detectable by conventional ultrasound techniques less than about 0.5 microns in size. Accordingly, there remains a desire for improved liposome compositions which may be efficiently produced and which have average particle sizes less than about 0.5 microns. Moreover, there remains a desire for improved liposome compositions which are efficiently produced, stable in vivo, and provide a higher degree of resolution upon acoustic imaging.
Thus, there exists a need in the art to provide further improvements in the use of liposome technology for the therapeutic and diagnostic administration of bioactive molecules. More specifically, there remains a desire in the art for improved methods for administration of amphipathic peptides including, but not limited to, members of the VIP/GRF family of peptides in liposomes in order to achieve a more prolonged and effective therapeutic effect.
The present invention provides improved methods of preparing biologically active liposome products comprising biologically active amphipathic compounds in association with a liposome. The liposomal formulations of the invention deliver and enhance bioactivity of the biologically active peptides in a manner which provides improvements in the efficacy and duration of the biological effects of the associated peptides. Increased efficacy and duration of the biological effect is believed to result, at least in part, from interaction of the compound with the liposome in such a manner that the compound attains, and is maintained in, an active or more active conformation than the compound in an aqueous environment. The invention thus overcomes the problems associated with previous liposomal formulations, such as, but not limited to, uptake by the reticuloendothelial system, degradation of the compound, or delivery of the compound in an inactive conformation.
According to one aspect of the invention, a method is provided for preparing a biologically active liposome product comprising a biologically active amphipathic compound in association with a liposome; said method comprising the steps of a) mixing a combination of lipids wherein said combination includes at least one lipid component covalently bonded to a water-soluble polymer; b) forming sterically stabilized liposomes from said combination of lipids; c) obtaining liposomes having an average diameter of less than about 300 nm; and d) incubating liposomes from step c) with a biologically active amphipathic compound under conditions in which said compound becomes associated with said liposomes from step c) in an active conformation. According to one aspect of the invention, the biologically active liposome products comprise unilamellar liposomes which are generally preferred for therapeutic uses. According to an alternative aspect of the invention, the biologically active liposome products comprise multivesicular liposomes and are produced according to a method comprising an additional step of forming multivesicular liposomes, preferably by carrying out the steps of sequentially dehydrating and rehydrating liposomes obtained in step c) with said biologically active compound. Despite any accepted meanings to the contrary, multivesicular, multilamellar, and oligolameilar liposomes are intended as liposomes containing multiple and irregular internal xe2x80x9ccompartmentsxe2x80x9d, not in an xe2x80x9conion-likexe2x80x9d configuration, but as set forth in FIG. 1. The multivesicular liposomes may be used in the therapeutic uses of the invention but are particularly useful in echogenic diagnostic methods of the invention in which they exhibit surprising echogenic properties.
As one aspect of the invention, the liposomes are sterically stabilized liposomes (SSL) which are produced from a combination of lipids which includes at least one lipid component covalently bonded to a water-soluble polymer. This water-soluble polymer, which is preferably polyethylene glycol (PEG), acts to sterically stabilize the resulting liposome against uptake by components of the reticuloendothelial system.
The methods of the invention are useful with any biologically active amphipathic compound which can thereby be stably maintained in an active conformation in association with or within the lipid bilayer of the liposome. Preferred amphipathic compounds include those characterized by having one or more xcex1- or xcfx80-helical domains in their biologically active conformation and particularly those in which polar and apolar residues are separated on opposite sides of the helix. Particularly preferred amphipathic compounds useful with the invention include any member of the vasoactive intestinal peptide (VIP)/growth hormone releasing factor (GRF) family of peptides which includes biologically active analogs thereof. The mammalian and non-mammalian VIP/GRF family of peptides includes functional analogs of VIP and GRF, peptide histidine isoleucine (PHI), peptide histidine methionine (PHM), growth hormone releasing factor (GRF), pituitary adenylate cyclase activating peptide (PACAP), secretin, and glucagon. Like VIP, other members of the VIP/GRF family of peptides, and biologically active analogues thereof, can form amphipathic helices wherein hydrophobic and hydrophilic domains of the peptide are segregated and the hydrophobic domain(s) is capable of binding lipid bilayers. The invention also contemplates receptor antagonists having enhanced bioactivity in association with liposomes prepared by a method of the invention. A particularly preferred peptide for use according to the invention is VIP. The biologically active peptide products of the invention may be utilized in a wide variety of therapeutic and diagnostic uses wherein it is desired to deliver a high level of biologically active compound or to detect targeted delivery of the liposome product as will be described below.
The invention further provides improved acoustic diagnostic products which have surprising acoustic reflectivity properties despite the fact that they are less than 1000 nm and even 300 nm in average diameter. The results using the liposomes less than 300 nm in diameter are particularly surprising in light of the teachings of the art that such liposomes should range from 0.8 to 3.0 microns in diameter. Specifically, the invention provides methods for the preparation and use of multilamellar diagnostic liposomes having an average diameter of less than about 1000 nm and particularly less than about 500 nm for improved imaging using acoustic reflectivity techniques. Herein, acoustic reflectivity, echo-reflectivity, and ultrasonic imaging are used with essentially the same meaning. The method of the invention comprises the steps of mixing a combination of lipids wherein at least one lipid component is conjugated to a water soluble polymer, forming and obtaining liposomes from the mixed combination of lipids, incubating the liposomes with a biologically active amphipathic compound, and forming multilamellar liposomes having an average diameter of about less than 1000 nm. According to a preferred embodiment of the invention, the multilamellar liposomes are formed by carrying out a lyophilization method. In a preferred embodiment, the liposomes first formed from the mixture of lipids have an average diameter of about less than 300 nm and in another embodiment these liposomes are obtained by extrusion. A preferred multilamellar liposome of the invention has an average diameter of about less than 800 nm, but most preferably has an average diameter of less than about 300 nm. In a preferred embodiment, the water soluble polymer is PEG. Biologically active compounds of the invention include those which are capable of forming xcex1- or xcfx80-helical domains and preferably are chosen from members of the VIP/GRF family of peptides. The most preferred biologically active compound of the invention is VIP. While not intending to be bound by any theory of their invention, it is believed that the incorporation of a water-soluble polymer such as PEG into the multivesicular liposomes may make them more capable of reflecting acoustic energy in spite of their relatively small size. While it is not completely understood why this might be the case, one possibility is that the presence of the water-soluble polymer acts to separate the walls of the multiple liposome vesicles making up a single multivesicular liposome and thus rendering that liposome better capable of reflecting acoustic radiation.
The invention further provides improved acoustic diagnostic methods wherein the small diagnostic liposomes of the invention comprising a biologically active amphipathic compound are administered in a diagnostically effective amount to a target tissue and the liposomes are detected in vivo using acoustic reflectivity. The preferred target tissue of the invention is a tumor. In one embodiment, the biologically active compound is characterized by having at least one or more xcex1- or xcfx80-helical domains. In a preferred embodiment, the compound is any member of the VIP/GRF family of peptides, and in a most preferred embodiment, the compound is VIP.